P. aeruginosa biofilms: RNA's role in biofilm stability

As an expert in biofilm exopolymer characterisation, Sudarsan Mugunthan , 29, has scored a Nature Communications publication in November 2023. His paper “RNA is a key component of extracellular DNA networks in Pseudomonas aeruginosa biofilms” (supervised by Prof Staffan Kjelleberg & A/Prof Thomas Seviour ) distilled new insights on biofilm stability, and means of targeting biofilm-specific traits for microbial control. Read on for more.

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Biofilm matrix

A biofilm – microbial cells embedded within a self-produced matrix – is the default mode of life for microorganisms that imparts significant advantages over a free-living lifestyle. The biofilm matrix is comprised of extracellular polymeric substances (EPS) that are diverse and vary according to the microbial species (e.g., bacterial pathogen).

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These EPS form complex structures that can restrict diffusion of antimicrobials and make bacteria highly resistant to external stresses, and hence stable. This could render both the biofilm and its residing bacteria more pathogenic, especially in sites of chronic infection such as the lungs or urinary tract. Disrupting the matrix provides a means for microbial control, as cells become significantly more vulnerable to antimicrobial technologies when outside the protective EPS environment.

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EPS

Disrupting EPS structures is key to ensuring that unwanted biofilm does not form in both medical (e.g. harbouring pathogens) and industrial (e.g. obstructing water pipes) settings. Although extracellular DNA and RNA are recognised as key components of the EPS, their study has been limited by an inability to characterise the polymers for investigation, owing to a lack of appropriate analytical approaches.

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Research questions

1)????? How does EPS provide stability to biofilms?

EPS provide a protective environment for the embedded cells by shielding inhabitants from external stressors, such as antimicrobials (that cannot readily penetrate to the interior of the biofilm), and desiccation. Microbes embedded within a matrix are also held in close and consistent proximity to each other. This enables cooperative and coordinated behaviour for chemical signalling, sharing and cycling resources, as well as the ability to horizontally transfer genetic material, such as antimicrobial resistance genes. Collectively, these emergent properties impart biofilm-based microbes with a resilience that fosters stability for the microbial community as a whole. This does not occur for free-living (planktonic) microbial assemblages.

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2)????? Does any specific EPS structure contribute to biofilm stability?

Different exopolymers within the EPS perform different roles within the matrix that ultimately affect stability. For example, polysaccharides have been implicated in elasticity, while specific proteins act as a framework builder, and nucleic acids can perform structural roles. Collectively, the different EPS components contribute to biofilm establishment, performance and stability, through a variety of mechanisms.

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RNA is key

At SCELSE, we tackle the above questions by using biophysical, microbiological, and biochemical approaches to identify and characterise EPS in P. aeruginosa biofilms. The uniqueness of this project lies in its ability to assign new functions to extracellular nucleic acid polymers, both DNA and RNA, elucidating both structural and functional aspects of the biofilm. These new insights were enabled by coordinated multidisciplinary approaches. This yielded a greater understanding of biofilm stability, as well as means of targeting biofilm-specific traits for microbial control.

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Prior to our study, RNA was not understood to be a structural component of the matrix as it was considered a biproduct of cell lysis that was unstable in the environment. Our study refutes this paradigm, demonstrating eRNA’s role as a key component of extracellular DNA networks. This finding will open up new opportunities for controlling biofilms by disrupting the matrix EPS.

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For the first time ever, we have identified an extracellular role/function for RNA outside cells in biofilms. Our group at SCELSE has shown that P. aeruginosa biofilms secrete specific type of extracellular RNA that can coexist with extracellular DNA outside cells to phase separate into gel like material and form sticky, viscoelastic biofilms. This in turn can promote biofilm stability.

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Medical impact

We identified higher abundance and co-existence of lasB mRNA and eDNA collected from COPD (chronic obstructive pulmonary disease patients) lung infected sputum. This study offers information on the exact nature and structure of RNA and DNA in the biofilms and provide means for identifying individual RNA in the biofilm matrix.

With this information, we can design a much more targeted approach (such as developing enzymes specific for breaking DNA:RNA hybrid structures) to disrupt biofilms at infection sites such as cystic fibrosis (CF) patient’s lungs, which otherwise cannot be efficiently degraded using existing knowledge and technologies.

Possible biofilm control

1)????? We could use this information on nature of specific RNA species and their types of interactions with DNA from this study to carefully design a CRISPR-Cas system to knock out targeted RNA from biofilms.

2)????? Understanding extracellular nucleic acid interactions has informed on the use of a combination of enzymes to degrade RNA:DNA complex higher order structures in biofilms, thereby destabilising biofilm matrix integrity.

New microscopy method

We have employed, for the first time, a new microscopy method called single molecule inexpensive fluorescent hybridisation (smiFISH) to locate individual extracellular RNA transcripts in P. aeruginosa biofilms. This provides information on the presence and relative location of eRNA within the matrix, at a far greater resolution than previously possible.

Conclusion

This study is novel given that not much is known about the interaction of RNA with other components. It is the first time anyone has identified a role for RNA in biofilms and their structural stability. Further investigation is needed to unravel the interactions with other biofilm components, such as co-occurring EPS molecules or extracellular signalling systems.